Cancer is the most common cause of death in many parts of the world and over 2.5 million cases of cancer are diagnosed globally every year. Recent advances in our understanding of the molecular biology of cancer have shown that cancer is a genetic disease resulting in the abnormal proliferation of the affected cell. Therefore, cancer therapists are now focusing on therapeutic strategies that involve macromolecules carrying genetic information, rather than a therapeutic protein itself, allowing for the exogenously delivered genes to be expressed in the tumor environment. Gene therapy is believed to offer therapeutic benefits to cancer patients in a number of ways that are not possible with conventional approaches. Traditional small molecule drugs usually function by non-specific interaction with the cellular targets, produce undesirable side effects and do not treat the root cause of the disease. Protein drugs which have been introduced over the last several years have their own limitations due to their rapid degradation and high doses that are required which often leads to undesirable side effects. Gene therapy uses the body's own cellular machinery to produce sustained therapeutic levels of proteins in specific tissues and cells after a single injection, thus providing a safe and effective method of treatment with better patient compliance.
The most commonly applied cancer gene therapy strategies include immunotherapy, cell ablation and anti-angiogensis accomplished by 1) local, 2) loco-regional, or 3) systemic injection. Cancer immunotherapy is a potent approach to combat cancer by stimulating the immune system against the cancer cells. Immunocytokines play an important role in the development of the host immune response by activation, maturation and differentiation of the immune cells. Several cytokines have been tested against a variety of cancers in human and in animal models of cancers. See Hum Gene Ther., 1998, vol. 9, 2223; Gene Ther. 1999, vol. 6, 833; Cancer Gene Ther. 2000, vol. 7, 1156; J. Control Rel. 2003, vol. 87, 177; and Cancer Res., 2002, vol. 62, 4023. Interleukin 12 (IL-12) is an immunostimulatory cytokine that shows great promise in the treatment of human cancer. See The Oncologist, 1996, vol. 1, 88. IL-12 is a 70-kD heterodimer consisting of two covalently linked chains, p35 and p40. The biological effects of IL-12 include the induction of IFN-γ production both by resting and activated CD4+ T cells, CD8+ T cells, and natural killer (NK) cells. IL-12 also enhances the proliferation of activated T and NK cells, increases the lytic activity of NK/lymphokine-activated killer cells, and facilitates specific cytotoxic T lymphocyte (CTL) responses.
In animal models, recombinant IL-12 has been demonstrated to induce profound T-cell mediated antitumor effects causing regression of established tumors, followed by systemic immune memory. See The Oncologist, 1996, vol. 1, 88. However, systemic administration of recombinant IL-12 has resulted in dose limiting toxicity in several experimental trials and in an initial human trial. See Lab Invest., 1994, vol. 71, 862; Science, 1995, vol. 270, 908; J. Interferon Cytokine Res., 1995, vol. 14, 335. Dose limiting toxicity was also observed with intraperitoneal administration of recombinant IL-12 in a recent human clinical trial. Clin. Cancer Res., 2002, vol. 8, 3686. A gene delivery approach that can provide therapeutic levels of IL-12 locally at the tumor site would have the advantage of generating an anticancer response without causing systemic toxicity.
Both viral and non-viral gene delivery systems have been used for IL-12 gene delivery in animal models of cancer. The viral approach has serious practical limitations due to toxicity concerns mainly because of an increased incidence of cancer and a strong immune reaction to viral antigens by the host system. There is considerable interest in the development of non-viral gene delivery systems due to their lesser toxicity. Using polyvinylpyrrolidone (PVP), a non-viral gene delivery system, for the delivery of IL-12 to treat renal carcinoma (Renca) and colon cell carcinoma (CT26) has been demonstrated. See Gene Ther., 1999, vol. 6, 833. When tumors were subjected to this gene therapy, they displayed all the characteristics of IL-12 protein therapy, e.g., an increased infiltration of NK cells, CD4 and CD8 T cells, coupled with an increased expression of major histocompatibility complex (MHC) class I molecules. IL-12 gene delivery was well tolerated and highly effective against both Renca and CT26 tumor bearing animals. Tumor rejecting mice were also protected from a subsequent rechallenge, suggesting the presence of a long lasting systemic immunity. A functionalized and less toxic water soluble lipopolymer (WSLP) has been tested for delivery of the IL-12 gene to CT26 colon carcinoma tumors. See Mahato et al, Mol. Ther., 2001, vol. 4, 130. IL-12 plasmid (pIL-12) and WSLP (pIL-12/WSLP) treatment gave higher levels of intratumoral gene expression than naked DNA.
Furthermore, secondary effects of the cytokine IL-12 production, namely IFN-γ and nitric oxide (NO) levels were also higher in WSLP treated tumors when compared with naked DNA. A single injection of pIL-12/WSLP complexes produced suboptimal effects on tumor growth and animal survival, while repeated delivery yielded better efficacy which indicates insufficient delivery by the system. J. Control Release 2003, vol. 87, 177. Similarly, intratumoral injection of IL-12 plasmid in another polymeric carrier, PAGA, produced only partial inhibition of CT26 tumors. See Gene Ther., 2002, vol. 9, 1075. These results warrant the need for more efficient delivery systems. Despite their insufficiencies in earlier preclinical trials, the excellent molecular flexibility of polymeric gene carriers allows for complex modification and novel functionalization imperative for the development of more efficient gene delivery systems.
It is widely recognized that employing a single treatment strategy against cancer is generally ineffective due to the multi-factorial nature of this disease. The combination of more than one drug to maximize the anticancer response is being increasingly utilized. See Gene Ther., 2000, vol. 11, 1852. It has been demonstrated that there is a synergistic relationship between IL-12 gene therapy and IFN-α gene therapy. Co-treatment of Renca tumors with these two genes led to 100% tumor rejection which was higher than that achieved by treatments with either IL-12 (58%) or IFN-α (25%) alone. Similarly, CT26 tumors showed a 50% rejection rate with combination gene therapy which was higher than the 17% and 0% rejection rate achieved from single treatments of IL-12 and IFN-α, respectively. Tumors treated by combination therapy showed increased tumor-infiltration of NK and CD8 T cells when compared to tumors treated by single gene therapy. Gene transfer of methylguanine-DNA-methyltransferase (MGMT) into stem cells alongside with chemotherapy protected normal cells from chemotherapy and reduced chemotherapy systemic toxicity. Nature Reviews Cancer 2004, vol. 4, 296.
Furthermore, combination gene therapy increased the number of CD40 molecules on antigen presenting cells (APCs) in the tumors to levels higher than was achieved with single treatments. Increased upregulation of CD40 on APCs is associated with higher activation status for antigen presentation. See Nature, 1998, vol. 393, 480; Nature, 1998, vol. 393, 474; and Nature, 1998, vol. 393, 478. A similar increase was observed in the levels of mRNA for the chemokines IP-10 and TCA-3. Combination gene therapy therefore synergistically enhanced the anti-tumor immunity and this effect was found to be long lasting in tumor rechallenge studies. Similar combination gene therapy studies have been reported by other groups. See Laryngoscope 2001, vol. 111, 815. Established tumors were treated with pIFN-α/PVP, pIL-2/lipid, or pIL-12/PVP alone or a combination thereof. The pIFN-α/PVP combination compared with the other two therapies significantly increased the antitumor effects when compared with single treatments. In another study utilizing the same tumor model, it has been demonstrated that combined treatment with pIL-12/PVP and pIL-2/lipid gave significantly higher anti-tumor effects when compared with single treatments. See Arch. Otolaryngol Head Neck Surg, 2001, vol. 127, 1319.
In another study, intratumoral injection of polyplexes of linear polyethylenimine (PEI) with an anti-oncogene and somatostatin receptor subtype 2 (sst2), produced a significant inhibition of growth of pancreatic tumors and metastases to the liver. Curr Opin Biotechnol, 2002, vol. 13, 128. The PEI-mediated delivery of sst2 in tumors led to increased apoptosis and activation of the caspase-3 and poly(ADP-ribose) pathways. Sustained delivery of DNA/PEI polyplexes into solid tumors produced higher expression than achieved by bolus delivery. Gene Ther., 1999, vol. 10, 1659. Dendrimers were used for inhibition of pancreatic carcinoma and hepatocellular carcinoma by intratumoral gene transfer of Fas-L and HSV-1 thymidine kinase, respectively. See Gene Ther., 2003, vol. 10, 434; and Gene Ther., 2000, vol. 7, 53.
Chemo-immunotherapy using cytotoxic drugs and cytokines offers a new approach for improving the treatment of neoplastic diseases. The therapeutic efficacy of combinations of IL-12 proteins with cyclophosphamide, paclitaxel, cisplatin or doxorubicin has been investigated in the murine L1210 leukemia model. See Int. J. Cancer, 1998, vol. 77, 720. Treatment of L1210 leukemia with IL-12 or one of the above chemotherapeutic agents given alone resulted in moderate antileukemic effects. Combination of IL-12 with cyclophosphamide or paclitaxel produced no augmentation of antileukemic effects in comparison with these agents given alone. However, combination of IL-12 with doxorubicin augmented the antileukemic effect, while combination with cisplatin had a moderate enhancing effect.
However, in murine melanoma MmB16 model the IL-12+paclitaxel combination was more effective than the individual therapies. Cancer Lett., 1999, vol. 147, 67. The antitumor efficacy of IL-12 protein in combination with adriamycin, cyclophosphamide, or 5-FU in MB-49 bladder carcinoma and B16 melanoma has also been examined. See, Clin. Cancer Res., 1997, vol. 3, 1661. In combination with chemotherapy, IL-12 administration increased antitumor activity without causing additional toxicity. In mouse sarcoma MCA207 that is refractory to treatment by either IL-12 or cyclophosphamide, combination of recombinant IL-12 and cyclophosphamide gave a better antitumor response than the individual treatments. J Immunol., 1998, vol. 160, 1369. In mouse mammary tumors, combination therapy comprising intravenous paclitaxil chemotherapy and intratumoral IL-12 gene therapy (IL-12/WSLP) was more efficacious than the individual therapies. See, Molecular Therapy, 2004, vol. 9, 829. The benefit of this combination therapy was dependent on the delivery vehicle used for paclitaxel. The synergistic interaction between paclitaxel and IL-12 gene therapy was observed when paclitaxel was formulated in a polymeric formulation. In comparison, combination with Cremophor EL (Taxol®), a widely used paclitaxel formulation for cancer therapy, was not synergistic, suggesting that the observed benefits were formulation specific.
To achieve a desirable outcome from a combination approach involving gene therapeutics, the selection of an appropriate gene delivery system is important. The gene delivery system used in the aforementioned combination experiments (Molecular Therapy, 2004, vol. 9, 829) is a water soluble lipopolymer, PEI-Cholesterol (WSLP). In the present invention, we describe the use of a novel class of polymeric carriers (PEG-PEI-Cholesterol) structurally distinct from WSLP in that it contains a hydrophilic polymer designed to improve pharmacokinetics, safety and potency of the gene delivery system and membrane interacting ligands (e.g., cholesterol) that are oriented in numerous geometrical configurations to promote transfection activity of anticancer genes either alone or in combination with a chemotherapeutic agent. The transfection activity advantage of PPC compared to WSLP in tumor tissue is illustrated in FIG. 1 and FIG. 2.
The combination of either two chemotherapeutic agents or a chemotherapeutic agent and a cytokine has been examined clinically. Although these combinations have produced greater tumor regression, the long-range survival benefits are marginal and cytotoxicity has been a problem. This is due to the inherent systemic toxicity associated with chemotherapy and recombinant protein therapy. New and more effective combinational approaches must be designed to improve future cancer therapy. In this present invention, we describe a novel combinational approach for treatment of cancer comprising a nucleic acid based therapeutic delivered with a polymeric carrier and at least one chemotherapeutic agent.